The present invention relates to a still-picture acquisition method and apparatus applied to a microscope and, more particularly, to a still-picture acquisition method and apparatus applied to a microscope, which are used in observing the biodynamics of a sample such as a living cell by using a fluorescence probe, and pick up images of a living cell or the like that shows a dynamic behavior and acquire its still picture.
Recently have been made attempts to visualize the ion concentration, film potential and so forth by means of a fluorescence probe using optical microscopes. For instance, the biodynamics of samples such as a neurocyte, particularly, the biodynamic behaviors thereof, are observed.
While such observation of a neurocyte or the like is intended to observe the dynamic behavior of a cell, it is effective to photograph one process of the dynamic behavior to clearly record the process as a still picture.
Catching one process of the dynamic behavior of a neurocyte or the like, however, requires the time resolution of a millisecond order so that the exposure time in photographing such a process becomes shorter to approximately a millisecond.
According to the conventional still-picture acquisition apparatus for the microscope that uses a photosensitive material such as a silver film, therefore, the photosensitive material suffers an insufficient sensitivity. Further, as the sensitivity is increased, the graininess becomes degraded. It has therefore been impossible to photograph a fluorescence-marked cell under a fluorescence microscope in such short exposure.
An apparatus and method which observe the dynamic behavior of an organic sample using a solid-state image pickup device or an optical scan type microscope are disclosed in, for example, Jpn. Pat. Appln. KOKAI Publication No. 5-219937. This publication describes an organic sample observing system using a solid-state image pickup device. This system is aims at making an observation over a long period of time to track the growth and proliferation of an organic sample and carries out so-called “intermittent photographing” to track the growth and proliferation of an organic sample over a time span of the order of several hours to several tens of hours while acquiring images at given time intervals. That is, the exposure and photographing timings are not determined by the dynamic behavior of a cell sample but merely take place at preset time intervals. This prior art is therefore inadequate for use in recording the images of the dynamic behavior of a living cell, such as a neurocyte, that varies at a high speed.
Jpn. Pat. Appln. KOKAI Publication No. 10-10436 discloses means which observes the physiological phenomenon of a cell immediately before and after application of a trigger signal using an optical scan type microscope. The disclosed technique stimulates a sample by means of a trigger signal which is obtained by detecting light from the sample and acquires the image of the sample immediately after the stimulation.
Because this prior art system acquires only the (one) image of a sample immediately after (or immediately before) stimulating the sample, however, the amount of fluorescence photons that can be sensed per pixel in a short time (about a millisecond) during which an image is acquired is reduced to the order of several to several tens of photons particularly in an application of measuring a fast phenomenon. Even if the specifications and performance of an electric circuit, such as the light-receiving sensitivity and SN ratio of a fluorescence sensor or probe, are adequate, therefore, it is inevitable from the viewpoint of the quantum physics that random noise caused by the quantum noise that is determined by the square root of the number of photons is superimposed on an acquired image, thereby degrading the image quality. While this prior art provides means for observing the dynamic behavior of a cell using an optical scan type microscope, it has no countermeasure against the degrading of the image quality caused by the quantum noise that increases as the sensing speed increases. The prior art system is therefore inadequate for usage in showing the dynamic behavior of a fast-varying living cell, such as a neurocyte, as high-quality still pictures.
A rotary disk scanner which simultaneously scans with multiple light beams is more suitable for fast image pickup to observe the dynamic behavior of a neurocyte than the scanner that scans with a single light beam as described in the Jpn. Pat. Appln. KOKAI Publication No. 10-10436.
A system which uses the rotary disk scanner that simultaneously scans with multiple light beams should synchronize the exposure time for photographing with the disk rotation at the time of picking up a high-quality still picture using the rotary disk scanner, but assumes no synchronization with the behavior of the cell sample. This system is therefore inadequate for usage in recording the dynamic behavior of a living cell, such as a neurocyte, as high-quality still pictures.
None of the prior art techniques cannot acquire the dynamic behavior of a fast-varying living cell, such as a neurocyte, as high-quality still pictures.